The maintenance of pH in liquid systems is extremely important to the operation and integrity of such systems, particularly where there is continuous generation of hydrogen ions due to activity in such systems. This activity may manifest itself in the form of chemical reactions, in the form addition of hydrogen ion to the systems from an external source, or from the environmental context of such systems, for example the introduction into such systems of hydrogen ions by dissolution or leaching. Many inorganic reactions produce hydrogen ions as a by-product, or are pH sensitive. In the area of pollution control, for example, many waste effluents are particularly characterized by having a low pH which must be neutralized without the addition of other soluble cations such as sodium which in turn are pollutants. Thus, neutralization of acidic pickling liquors by use of sodium hydroxide results in a waste stream containing substantial quantities of sodium sulfate which cannot be disposed of because of the high dissolved sodium sulfate solids content.
Similarly, biochemical reactions and functions of biological systems are pH dependent. There is a particular pH for every enzyme at which its activity is optimal; there are specific pH ranges in which an organism will grow and others in which it may synthesize certain molecules; and there are specific pH ranges in which a cell must be kept in order for it to maintain itself.
At present, control of pH in biological reactions in vitro is achieved with the use of chemical buffers having pK values in the range 6.5 to 8.0. Of the limited number of such buffer systems available, only the phosphates and carbonates are non-toxic. However, even these two buffers present problems for pH maintenance of cellular systems. For example, at the concentration of phosphate required to maintain pH in the system of stored red blood cells, the cells undergo tremendous osmotic stress due to the redistribution of water across the cell membrane in response to the difference in phosphate ion concentration inside and outside the cells. Further, the buffering capacity of the phosphate is lost at the temperatures required for storage (preservation) of these cells. As for the carbonate systems, their pK values allow them to buffer only at the extremes of the physiological range (i.e., pH 6.5 and 8.0). Recently, Harmening and Dawson (The Use of Ion Exchange Resins as a Blood Preservative System, 30th Annual Meeting of the American Association of Blood Banks, Atlanta 1977) have proposed use of ion exchange resins to control pH in some biological systems in vitro. However, such materials tend to decompose on standing, permit toxic monomers or plasticisers to leach out, or may bind essential metal ions, all of which become significant problems for the maintenance of biological systems.
The term fermentation refers to the controlled synthesis of biologicals (e.g., antibiotics, vitamins, steroids) by micro-organisms in aerobic as well as anaerobic processes. The present methods used in industrial fermentation for pH control are:
(1) constant monitoring of pH by glass electrode and titration with required acid or base to regain desired pH; PA1 (2) use of various buffers such as phosphates, carbonates, Tris, etc.
Nearly all fermentation reactions are carried out in the pH range 5 to 10. Production of low molecular weight molecules by micro-organisms is usually carried out in the pH range 5 to 7, such molecules being acetone, butanol and ethanol. Larger molecules such as antibiotics and steriods are produced by micro-organisms in culture at pH 7.0 to 7.5. A general discussion of industrial fermentation processes and pH requirements may be found in Biochemical and Biological Engineering Science, Vol. 1, N. Blakebrough, editor, Academic Press, London (1967).
Penicillin fermentation is an example where maintenance of pH in the range 7.0 to 7.4 is required in order to get the micro-organisms (i.e., penicillium notatum and penicillium chrysogenum) to produce the antibiotic. Caden has reported that even though growth of the penicillin molds is facilitated in the pH range 4.5 to 5.0 the production of penicillin by these micro-organisms required the media pH to be in the range of 7.0 to 7.5 (E. L. Caden, J. Biochem. Microbiol. Tech. Engng. 1, 413 (1959). Brown and Peterson have reported that the optimum pH for production of penicillin for prolonged period is 7.0 (W. E. Brown and W. H. Peterson, Industr. Engng. Chem. 42, 1769 (1950). Pirt and Callow recommend a pH below 7.0 for growth of the penicillin mold (Stage I), and a pH between 7.0 and 7.4 for production of penicillin by the mold (Stage II) (S. J. Pirt and D. S. Callow, Nature 184, 307 (1959).
The term "cells in culture" refers to any cells (single or connective) or collection of cells (e.g., organs) that are maintained to some degree in vitro in an aqueous media. The pH range required for storage and growth media used with in vitro maintenance of mamalian cells and organs is 7.0 to 7.4.
Although various "buffers" such as Tris, phosphate, N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid, and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, have been proposed for pH control of tissue culture systems they have not proved adequate for maintenance of normal cell activity. In part this problem arises from the need for bicarbonate in the media. The maintenance of a stable pH in the presence of the easily disturbed bicarbonate-CO.sub.2 equilibrium can frequently be a problem, the solution for which has been the use of relatively high levels of phosphate buffers (e.g., greater than 50 mM) or elaborate CO.sub.2 --HCO.sub.3.sup.- regulating equipment. A general discussion of environmental factors influencing cells in culture can be found in Growth, Nutrition and Metabolism of Cells in Culture, Vols. I and II, G. H. Rothblat and V. J. Cristofalo, editors, Academic Press, New York (1972).
There is also a great need to improve the viability of stored blood under more closely physiological conditions so that it is useful over longer periods of time. In 1977 ten to twelve million units of blood will have been collected in the U.S. At present, FDA regulations permit each of these units to be stored three weeks before they can no longer be considered for use for transfusion. This limit in permitted storage time contributes to a loss of collected blood ranging in degree from 20 to 50%. The cost of this loss, at $50.00 a unit, runs over one hundred million dollars a year.
In addition to the inventory, supply, and cost problems resulting from loss due to outdating, there is also the clinical problem of significant biochemical deterioration of the stored bank blood. After two weeks the stored blood has undergone significant biochemical deterioration which can result in a 30% loss in red cell survival after transfusion. The recipient patient also has the burden of "repair" of the deteriorated red cells received, this repair can take from 12 to 72 hours before the transfused blood is returned to a normal state. Such burden of repair may present serious problems to patients receiving large volumes of blood which put added medical strain on the patient. Those red cells that cannot be "repaired" represent an excretory problem and burden.
Thus, there is a great need not only for prolonging the storage life of blood in order to reduce loss due to outdating, but also to improve the quality and viability of blood stored longer than two weeks. Central to achieving the goals of longer shelf life and improved quality of bank blood is the need to continuously control the pH in blood in the narrow pH range of 7.0 to 7.2 while it is being stored in 4.degree. C. In addition to pH, the parameters considered most important to red cell quality are glucose utilization, cell morphology, cellular levels of ATP (adenosine triphosphate) and 2,3-DPG (2,3-diphosphoglycerate). Thus, an improved method of blood storage should maintain a more physiologically compatible pH level, with attendant improved results in maintaining better ATP and 2,3-DPG levels than standard CPD blood at 3 and 4 weeks of storage, and better morphology.
Accordingly, the importance of being able to maintain pH in the range 6.9 to 7.5 is far reaching in medicine, the pharmaceutical industry and the research laboratory. Equally important however is the ability to maintain the pH in an non-interfering, nontoxic and continuous fashion. There is thus a great need for an improved method and apparatus for continuous control of pH in liquid systems, and more particularly in biological and biochemical systems. Important among these latter, there is a great need for improvement in the viability of stored blood during the presently accepted storage term of 21 days, and thereafter, and for improved anticoagulant solutions.